Use of the Antimicrobial Peptide Sublancin with Combined

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Use of the antimicrobial peptide sublancin with combined antibacterial and immunomodulatory activities to protect against methicillin-resistant Staphylococcus aureus infection in mice Shuai Wang, Qingwei Wang, Xiangfang Zeng, Qianhong Ye, Shuo Huang, Haitao Yu, Tianren Yang, and Shiyan Qiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02592 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 16, 2017

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Journal of Agricultural and Food Chemistry

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Use of the antimicrobial peptide sublancin with combined antibacterial and

2

immunomodulatory activities to protect against methicillin-resistant

3

Staphylococcus aureus infection in mice

4 5

Shuai Wang, †,‡ Qingwei Wang, † Xiangfang Zeng,† Qianhong Ye,† Shuo Huang,†

6

Haitao Yu,† Tianren Yang† and Shiyan Qiao*,†

7 8

†

9

100193, China

State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing

10

‡

11

Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China

Department of Animal Nutrition and Feed Science, College of Animal Science &

1

ACS Paragon Plus Environment


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ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is the major

13

pathogen causing serious hospital infections worldwide. With the emergence and

14

rapid spread of drug-resistant bacteria, there is extraordinary interest in antimicrobial

15

peptides (AMPs) as promising candidates for the treatment of antibiotic-resistant

16

bacterial infections. Sublancin, a glycosylated AMP produced by Bacillus subtilis 168,

17

has been reported to possess protective activity against bacterial infection. The present

18

study was performed to evaluate the efficacy of sublancin in prevention of MRSA

19

ATCC43300 intraperitoneal infection in mice. We determined that sublancin had a

20

minimum inhibitory concentration of 15 µM against MRSA ATCC43300. The

21

antimicrobial action of sublancin involved the destruction of the bacterial cell wall.

22

Dosing of mice with sublancin greatly alleviated (p < 0.05) the bacterial burden

23

caused by MRSA intraperitoneal infection as well as considerably reduced the

24

mortality and weight loss (MRSA vs 2.0 mg/kg sublancin on day 3: 19.2 ± 0.62 g vs

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20.6 ± 0.63 g) of MRSA challenged mice (p < 0.05). Sublancin was further found to

26

balance the immune response during infection and relieve intestinal inflammation

27

through inhibition of NF-κB activation (p < 0.01). Taken together, with combined

28

antibacterial and immunomodulatory activities, sublancin may have potent therapeutic

29

potential for drug-resistant infections and sepsis.

30

KEYWORDS:

31

Immunomodulatory, Mice

Antimicrobial

peptide,

Staphylococcus

2


aureus,

Sublancin,

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INTRODUCTION

33

Staphylococcus aureus is one of the most common human pathogens and can cause

34

various often difficult-to-treat human illnesses ranging from minor skin abscesses to

35

life-threatening infections, such as pneumonia, endocarditis, pseudomembranous

36

enteritis, bacteremia and sepsis.1,2 Additionally, S. aureus is also a major pathogen

37

responsible for contamination of a broad variety of foods.3 Cases of foodborne

38

illnesses caused by microbial contamination are increasing in many countries

39

(185,000 cases in the United States each year) and methicillin-resistant S. aureus

40

(MRSA) strains have become a global concern.4-6 This has been aggravated by a

41

collapse in the number of approvals of new antibacterial agents in the past three

42

decades.7

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Antimicrobial peptides (AMPs) are a variety of naturally occurring molecules

44

that provide immediately effective and non-specific defenses against invading

45

pathogens.8 AMPs have been considered potential alternatives to conventional

46

antibiotics to treat drug-resistant bacteria, and a few AMPs are being tested in clinical

47

trials.9, 10 Sublancin is a 37-amino acid AMP isolated from Bacillus subtilis 168 with

48

high stability.11 It exhibits bactericidal activity against several species of

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Gram-positive bacteria, such as pathogenic strains S. aureus and Streptococcus

50

pyogenes.11 On the other hand, the gene, named yolF, is crucial for immunity of B.

51

subtilis itself to sublancin.12 Numerous studies have investigated the antimicrobial

52

effects of sublancin against drug-susceptible bacteria in vitro.11,13 However, the in vivo

53

efficacy against drug-resistant bacteria, the toxicity and the immunomodulatory 3



54

properties of sublancin have not been thoroughly investigated. In addition, naturally

55

occurring AMPs have not been successfully translated for clinical applications

56

because of unwanted toxicity, poor efficacy in vivo and high manufacturing costs.10

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Our previous study indicated that the minimum inhibitory concentration (MIC) of

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sublancin against Clostridium perfringens was much higher than that of lincomycin in

59

vitro, whereas it was needed in much smaller amounts to control necrotic enteritis

60

induced by C. perfringens in broilers than lincomycin.14 In addition, we found that the

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therapeutic efficacy of sublancin on S. aureus challenged mice was comparable with

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ampicillin, although the MIC of sublancin against S. aureus was higher than

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ampicillin.15 Emerging evidence indicates that AMPs can confer protection by

64

immune modulation in addition to direct destruction of microbes.16, 17 Therefore, we

65

hypothesize that sublancin possesses immunomodulatory properties.

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The aims of this study were to (i) evaluate the in vitro antibacterial activity of

67

sublancin against MRSA ATCC43300, (ii) assess the cytotoxic effects of sublancin

68

against murine macrophages and human colonic epithelial cells, (iii) investigate the

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protective effect of sublancin in a mouse sepsis model and explore the underlying

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mechanism of action of this peptide. The peptide therapy described here represents an

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innovative approach to treat infectious diseases depending on its combined

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antibacterial and immunomodulatory properties.

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MATERIALS AND METHODS

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Bacterial Strains, Culture Conditions and Chemicals. Methicillin-resistant S.

4


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aureus ATCC43300 was the test organism used throughout this study. This particular

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MRSA strain has been widely used for assessing antibacterial activities of

77

antimicrobial substances or establishment of several animal infection models.18-20

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The bacteria were grown in LB broth at 37oC with 120 rpm shaking for 18 h.

79

Sublancin was generated in our laboratory using a highly efficient expression system

80

involving B. subtilis 800 as described previously.15 The amino acid sequence of

81

sublancin was GLGKAQCAALWLQCASGGTIGCGGGAVACQNYRQFCR and the

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relative molecular mass was 3879.8 Da. The peptide purity was above 99.6% as

83

determined by high performance liquid chromatography. Sublancin was produced as

84

lyophilized powder and stored at -20oC.

85

Assay of Antibacterial Activity. The antibacterial activity of sublancin was

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determined by the Agarose Diffusion Method as previously described.21 Molten LB

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medium containing 1% agar at 45oC was inoculated with an 18-h culture of MRSA

88

ATCC43300 to attain a final concentration of approximately 104 to 105 bacteria per ml,

89

and then poured into a 90 × 15 mm Petri dish. Upon solidification of the agar medium,

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the agar was perforated using a sterilized cork borer to make wells (8 mm in diameter).

91

Sublancin powder was dissolved in sterile water to a final concentration of 60 µM.

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Aliquots of 200 µl test samples were transferred into the 8-mm wells. The plate was

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pre-incubated at room temperature for 1 h, and then incubated at 37oC for 18 h. A

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negative control using water was also included.

95

Determination of Minimum Inhibitory Concentration. The MIC was

5



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evaluated by the broth microdilution technique in accordance with Clinical and

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Laboratory Standards Institute (CLSI) guidelines.22 Briefly, sublancin was initially

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dissolved in distilled water, and serial two-fold dilutions were made in Mueller Hinton

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Broth using 96-well microplates. The concentration of sublancin ranged from 0.12 to

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60 µM. Ten microliters of MRSA ATCC43300 overnight broth culture was inoculated

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into each well with a concentration of 1.0 × 105 CFU/ml. The microtiter plate was

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incubated at 37oC for 24 h and bacterial growth was measured by a change in

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absorbance at 600 nm using a microplate auto reader. Positive (media with inoculum)

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and negative controls (media only) were included. The MIC was determined as the

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lowest concentration of sublancin that inhibited MRSA ATCC43300 growth (lack of

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increase in absorbance reading). The analyses were performed in triplicate.

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Time-kill Curves. MRSA ATCC43300 was grown in Mueller Hinton Broth

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containing a range of concentrations of sublancin (0, 0.125, 0.25, 0.5 and 1.0 × MIC).

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All cultures were incubated at 37oC in a 120-rpm shaker bath. Sampling times

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included 0, 0.5, 1, 2, 3, 4 and 24 h. Samples (100 µl) were removed from the tube and

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diluted 1:10 in sterile saline solution, and the solutions were subsequently plated onto

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Tryptone Soya Agar Plates (Oxoid, Basingstoke, Hampshire, England). Plates were

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incubated at 37oC for 24 h. Assays were performed in triplicate.

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Electron Microscopy. To further explore the morphology and ultrastructure

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changes in MRSA cells, Scanning (SEM) and Transmission Electron Microscopy

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(TEM) were performed. Two milliliters of MRSA ATCC43300 broth culture in the

6


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absence or presence of 0.25 × MIC sublancin were prepared as described in the

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time-kill assay and were collected at 0, 0.5, 1, 2, 3, 4 and 24 h, and centrifuged at

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3,000 × g for 10 min. The bacterial pellets were washed twice in PBS, and

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resuspended in 500 µl of PBS supplemented with 2.5% fresh glutaraldehyde for 1 h at

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room

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phosphate-buffered osmium tetroxide and processed for Scanning and Transmission

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Electron Microscopy by the methods previously described.23 Changes in cell

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morphology were observed by SEM. Up to 12 images of TEM for each time point

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were collected at 40,000 × magnification, and cells were observed for evidence of

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septal components. The percentages of MRSA cells containing septal components

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were calculated.

temperature.

Thereafter,

the

bacterial

pellets

were

fixed

in

1%

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Cell Cultures and Cytotoxicity Assays. The murine macrophage-like cells

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RAW264.7 was purchased from National Infrastructure of Cell Line Resource

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(Beijing, China) and maintained in Dulbecco’s Modified Eagle Medium (DMEM)

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supplemented with 10% fetal bovine serum (FBS) (Life Technologies). Peritoneal

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macrophages were isolated from BALB/c mice as previously described24 and

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maintained

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penicillin/streptomycin). The human colon carcinoma cell line Caco-2 was attained

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from the American Type Culture Collection (Rockville, MD, USA) and grown in

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DMEM supplemented 10% FBS, 100 U/ml penicillin/streptomycin, and 1%

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Non-Essential Amino Acids (NEAA) (Life Technologies). Cellular cytotoxicity of

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sublancin was determined using Cell Counting Kit (CCK-8) purchased from

in

supplemented

DMEM

(10%

FBS

7


and

100

U/ml


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Sigma-Aldrich (St. Louis, Mo. USA). This assay is based on the conversion of

140

water-soluble tetrazolium salt (WST-8) to a water-soluble formazan dye upon

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reduction via dehydrogenases in cells. The amount of the formazan dye generated by

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the activity of dehydrogenases in cells is proportional to the number of living cells.25

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RAW264.7 cells, mouse peritoneal macrophages, or Caco-2 cells were seed at a

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density of 2 × 104 per well in a 96-well plate and incubated at 37oC in a humidified

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atmosphere with 5% CO2 for 24 h. Then, cells were treated with sublancin at the

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indicated concentrations. Each concentration was repeated six wells. Twenty-four

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hours later, 10 µl of CCK-8 solution were added to each well and incubated at 37oC

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for an additional 2 h. The absorbance at 450 nm was determined with a microplate

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reader (Bio-Rad Laboratories, Hercules, CA). Cell viability was expressed as a

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percentage of the absorbance values in various concentrations of sublancin compared

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to that of the control (untreated) cells.

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In vivo MRSA Challenge Experiments. Six-week old female BALB/c mice

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were obtained from HFK Bioscience Co., Ltd. (Beijing, China). All mice used in this

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study were kept in plastic cages under 12 h light/dark cycle and had access to food

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and water ad libitum. Experiments on animals were performed in accordance with the

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Animal Care and Use Committee of China Agricultural University (Beijing, China).

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To study the anti-infective role of sublancin in an experimental model of

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MRSA-induced sub lethal infection, mice were challenged with MRSA ATCC43300

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(1.0 × 108 CFU/mouse) by intraperitoneal injection. After inoculation, mice were

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treated by intraperitoneally injection with 2.0 mg/kg body weight (BW) sublancin at

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the indicated time points (n = 10). Twenty-four hours after the bacterial challenge, the

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peritoneal fluid was collected from each mouse by lavaging with 1.5 ml of cold sterile

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saline. The staphylococcal load in the peritoneal lavage was enumerated as described

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previously.26 The protective effects of sublancin against MRSA and comparison of its

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therapeutic efficacy with traditional antibiotics was performed in a subsequent

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experiment. Mice were randomly allocated into one of four treatments (n = 12): (i)

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uninfected control, (ii) infected control, (iii) sublancin, and (iv) vancomycin.

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Vancomycin has been accepted as the first-line choice for treating infections due to

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MRSA.27 The mice were inoculated intraperitoneally with MRSA ATCC43300 (1.0 ×

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108 CFU/mouse), while mice in the uninfected control group were given the same

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volume of sterile saline. After inoculation, the mice received sublancin or vancomycin

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at the dose of 2.0 mg/kg BW via intraperitoneal injection at 6 h,15 whereas mice in the

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uninfected control and infected control groups were intraperitoneally injected with the

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same volume of sterile saline. For bacterial load evaluation and cytokine analysis, six

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mice/group were euthanized 24 and 72 h after infection. Peritoneal lavage was

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collected in 1.5 ml of cold sterile saline.

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To assess the therapeutic efficacy of sublancin in a lethal infection model

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induced by MRSA, mice were given a lethal intraperitoneal dose of MRSA

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ATCC43300 (5.0 × 109 CFU/mouse) in 0.5 ml sterile saline.15,28 Six hours after

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bacterial injection (day 0), mice (n = 20) were treated by intraperitoneal injection with

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and without various doses of sublancin. An uninfected control (where mice were 9



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uninfected and untreated) was also included. Body weight of all mice was measured,

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and the mortality was monitored for 3 days after infection. On day 3, six mice/group

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were euthanized. The jejunum and spleen tissues were collected for further research.

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Determination of Cytokines in Peritoneal Lavage. Peritoneal lavage samples

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were centrifuged at 1,000 × g for 10 min to obtain cell-free samples and preserved at

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-80oC. Cytokine levels including TNF-α, IL-6 and MCP-1 were analyzed by ELISA

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using commercially available Quantikine Kit (Cusabiol Biotech Company, Wuhan,

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China).

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Histological Examination and Immunohistochemistry. For histopathology,

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samples of jejunum (1 cm) and spleen were aseptically excised and fixed in 4.0%

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paraformaldehyde and then embedded in paraffin. Subsequently, transverse sections

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were cut at 5 microns and stained with hematoxylin and eosin before evaluation for

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histomorphometry. Immunohistochemistry was performed as previously described.15

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Images were taken using a CK-40 microscope (Olympus, Tokyo, Japan).

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Western Blot Analysis. The frozen jejunum samples were homogenized in RIPA

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lysis buffer containing protease inhibitors (Applygen, Beijing, China). Protein

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concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher

199

Scientific, Rockford, IL). Samples of 30 µg of protein were electrophoresed on SDS

200

polyacrylamine gels and electrotransferred to PVDF membranes (Millipore).

201

Membranes were blocked with 1 × TBST containing 5% of BSA (Sigma-Aldrich, St

202

Louis, MO) for 2 h at room temperature. The membranes were incubated with

10


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corresponding primary antibodies (1:1000 dilution for overnight at 4oC) against

204

NF-κB (p65), iNOS and β-actin (Cell Signaling Technology, Boston, MA). After

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washing of membranes with 1 × TBST, membranes were incubated with the

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HRP-conjugated goat anti-rabbit IgG (Huaxingbio Biotechnology, Beijing, China) for

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1 h at room temperature. The chemifluorescene was detected with the Western Blot

208

Luminance Reagent (Applygen, Beijing, China) using an ImageQuant LAS 4000 mini

209

system (GE Healthcare Bio-sciences AB, Inc., Sweden), and quantified by a

210

gel-imaging system with Image Quant TL software (GE Healthcare Life Science).

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Statistical Analysis. Data are expressed as means ± SEM except for the data in

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Table1 which is presented as means ± SD. Statistical analysis of data was conducted

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with the statistical software SAS Version 9.2. For the percentage of MRSA cells

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containing septal components, Mann-Whitney test was performed to evaluate

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differences between the mean values of the control and sublancin treated group. The

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other data were analyzed by one-way ANOVA using GLM procedures. Statistical

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differences among treatments were determined using Student Newman Keuls Multiple

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Range Test. A P value < 0.05 was considered significant.

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RESULTS

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Antibacterial Activity of Sublancin. The antibacterial effect of sublancin was

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assessed by agar well diffusion method (Figure 1A). The clear inhibition zones were

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observed when sublancin (60 µM) was transferred into wells made on the solidified

11



223

agarose mixed with MRSA ATCC43300. This result indicated that sublancin with

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antibacterial activity was successfully acquired.

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Minimal Inhibitory Concentration. The antimicrobial peptide sublancin was

226

active against MRSA ATCC43300 at MIC of 15 µM. No growth was observed in

227

wells supplemented with 15 µM sublancin, and growth was evident in wells with

228

0.12–7.5 µM sublancin.

229

Time-kill Assay. The results of the time-kill curve experiment are shown in

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Figure 1B. After 1 h, the viable count after exposure to 0.125, 0.25, 0.5 and 1.0 × MIC

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of sublancin was reduced in a concentration and time-dependent manner compared to

232

the control. 1.0 × MIC of sublancin was highly active with bacteriostatic activity

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resulting in a 3.34 log reduction in CFU/ml at 24 h relative to the control.

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Electron Microscopy. Similar appearance of MRSA was seen in the untreated

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cells collected at different sampling times and, therefore, only scanning electron

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micrographs of samples collected at 24 h are presented here. The morphological

237

changes in the MRSA cells caused by treatment with sublancin at the 0.25 × MIC

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concentration were evaluated by SEM analysis (Figure 1C). The untreated cells that

239

were incubated in Mueller Hinton Broth for 24 h showed smooth cell surfaces and

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appeared to be healthy and intact (Figure 1C-1). The MRSA cells treated by sublancin

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for 24 h had adulterated morphology, where cell walls had irregularities and impaired

242

changes rendering wall ruptures and cellular lysis in some cases (Figure 1C-2).

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Consistent with the SEM analysis, in the TEM images (Figure 1D), the MRSA 12


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cells treated with sublancin for 24 h exhibited several changes, including cell wall

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fragmentation and cell lysis (Figure 1D-4). In addition, MRSA cultures exposed to

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0.25 × MIC sublancin for 1 h had a significantly higher percentage of cells containing

247

septa (p < 0.01) compared to untreated cells (Figure 1D-3). These increases were

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established by 1 h and persisted until 24 h (Figure 1E).

249

In Vitro Toxicity of Sublancin. The utilization of AMPs as therapeutic agents is

250

greatly reduced because of their cytotoxic activity toward mammalian cells, such as

251

magainin 2 and cecropin A.29,30 We determined the cytotoxic effects of sublancin on

252

RAW264.7 macrophage cells, mouse peritoneal macrophages, and human Caco-2

253

epithelial cells. Cell viability was assayed using CCK-8 test after 24 hours of

254

treatment with various sublancin concentrations. As shown in Figure 2, sublancin

255

showed no significant cytotoxic effects on cells in culture, even up to the

256

concentrations of 500 to 1,600 µM.

257

Efficacy of Sublancin in MRSA Infection Models. The efficacy of sublancin

258

was assessed in a mouse intraperitoneal infection model caused by MRSA

259

ATCC43300. We observed that sublancin decreased bacterial counts (Figure 3) and

260

mortality (Table 1) (p < 0.05). Sublancin was given by intraperitoneal injection before

261

(-24 h) to after (+6 h) the MRSA challenge, and similar efficacy was proved in all

262

cases (Figure 3A). Common bacterial resistance did not provide any protection

263

against peptide, as sublancin decreased the number of viable bacterial counts in the

264

peritoneal lavage 24 (p < 0.05) and 72 h (p = 0.09) after infection. Additionally, mice

13



265

treated with vancomycin had fewer viable bacterial counts in the peritoneal lavage

266

than MRSA infected mice 24 (p < 0.01) and 72 h (p = 0.07) after infection.

267

Next, we investigated whether sublancin treatment was beneficial for a mouse

268

lethal MRSA sepsis model. Mice were given a lethal intraperitoneal dose of MRSA

269

ATCC43300 (5.0 × 109 CFU/mouse) and sublancin was injected intraperitoneally 6

270

hours after bacterial challenge. Body weight of all mice and survival were monitored

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for 3 days. On day 1, all MSRA ATCC43300 infected control animals showed general

272

clinical symptoms of lethargy, ruffled fur, diarrhea and anorexia, and the clinical signs

273

of infection still persisted on days 2 and 3. The sublancin-treated animals showed less

274

severe symptoms on days 2 and 3. As shown in Table 1, no death occurred in the

275

uninfected control group during the experiment. However, the cumulative mortality of

276

the infected control group reached 65% by day 3. Treatment with the 4 sublancin

277

levels (0.5, 1.0, 2.0, and 4.0 mg/kg) decreased cumulative mortality to 35, 25, 15, and

278

10% respectively. All mice that were challenged with MRSA ATCC43300 exhibited a

279

significant decrease in BW immediately afterwards compared with uninfected control

280

mice (p < 0.01). The average body weight in the sublancin treatments of 2.0 and 4.0

281

mg/kg was significantly higher than that of the infected control on days 2 and 3.

282

Sublancin Modulated Cytokine Production In Vivo. As sublancin effectively

283

controls MRSA infection, the influence of sublancin on the immune response to

284

MRSA was examined. As shown in Figure 4, MRSA infection significantly increased

285

(p < 0.05) TNF-α, IL-6, and MCP-1 concentrations in the peritoneal fluid 24 and 72

14


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hours after infection. The IL-6 and MCP-1 content in sublancin treated group tended

287

to be higher (p < 0.1) than that of infected control group at 24 hours post-infection.

288

Interestingly, TNF-α, IL-6, and MCP-1 induction by MRSA was significantly

289

decreased by sublancin at 72 hours post-infection, similar to levels seen in mice

290

treated with vancomycin.

291

Pathology and Histological Analysis. To gain further insight into the efficiency

292

of sublancin in a lethal MRSA sepsis model, jejunal tissue and spleen were aseptically

293

isolated for histopathology. As shown in Figure 5A, the jejunal villi height was

294

decreased (p < 0.01) in the MRSA infected mice compared with the uninfected

295

control. Mice challenged with MRSA and treated with the 4 levels of sublancin (0.5,

296

1.0, 2.0, and 4.0 mg/kg) also had greater (p < 0.01) jejunal villi height compared with

297

the infected control. To investigate the influence of sublancin on intestinal cell

298

proliferation, proliferating cell nuclear antigen (PCNA) was tested by immunostaining.

299

No data of the PCNA+ cells is shown for the MRSA group because of the intestine in

300

this treatment was seriously damaged and we could not acquire an exact cell count

301

(Figure 5B). However, the sublancin treatment (4.0 mg/kg) resulted in significant

302

increase (p < 0.05) in the number of PCNA+ cells than the uninfected control (Figure

303

5C).

304

Caspase-3 is an apoptotic marker, and it has been reported that S. aureus could

305

escape innate defenses and elicit the caspase-3 mediated immune cell death.31

306

Caspase-3 activation was observed in the spleen of MRSA infected mice, while less

15



307

caspase-3 staining was observed in the spleen from the 4.0 mg/kg sublancin treated

308

mice (Figure 6B). The CD4+ and CD8+ subsets of T lymphocytes are primarily

309

involved in the immune responses to specific antigenic challenges. We found that

310

MRSA challenge increased CD4+ T cell density in the spleen but had no significant

311

impact on CD8+ T cells (Figure 6C and D). The sublancin treatments (2.0 and 4.0

312

mg/kg) decreased the number of CD4+ T cells compared with the uninfected control.

313

Additionally, mice treated with sublancin exhibited an increase in the densities of

314

CD8+ T lymphocytes.

315

Sublancin Inhibited NF-κB Nuclear Translocation and iNOS Expression

316

Induced by MRSA. NF-κB plays a key role in regulating immune and inflammatory

317

processes.32 MRSA challenge significantly increased (p < 0.01) the expression of

318

NF-κB compared with uninfected control. Mice treated with sublancin had

319

significantly decreased (p < 0.01) NF-κB production than mice in the MRSA group

320

(Figure 7A and B). Activation of NF-κB is an essential step for iNOS expression,

321

which plays a crucial role in a variety of pathological processes including

322

inflammation.33,

323

iNOS expression. As indicated in Figure 7A and C, the relative expression of iNOS in

324

the MRSA infected control mice was greater (p < 0.01) than the uninfected control

325

mice. There was also evidence for decreased (p < 0.01) iNOS expression in mice

326

treated with sublancin.

327

DISCUSSION

34

We investigated the influence of sublancin on MRSA induced

16


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328

Antimicrobial peptides, which are widely distributed throughout the plant and animal

329

kingdoms, have been described as evolutionarily ancient weapons.35 The rapid

330

emergence of drug resistance in S. aureus along with the dwindling supply of

331

antibiotics that are effective against these pathogens has promoted an interest in

332

AMPs for potential therapeutic applications.36 Sublancin is a glycosylated AMP

333

known as glycocins.11, 37 In the in vitro study, both the clear zones of inhibition and

334

SEM demonstrated that sublancin showed growth inhibition against MRSA

335

ATCC43300. Consistent with a previous report, S. aureus CVCC1882 was susceptible

336

to sublancin as reflected by a MIC of 4.36 µg/ml.15 The time-kill assays demonstrated

337

reductions in viable cells by sublancin at concentrations ≥ 0.25 × MIC. Hence, we

338

choose 0.25 × MIC concentration for the electron microscopy analysis.

339

Presumably sublancin acts by forming pores in the microbial cell membrane of a

340

sensitive microbe.38 Depending on the specific AMPs studied, most manifest their

341

antibacterial activity by directly disrupting the bacterial cell membrane.39 In

342

agreement with this hypothesis, it has been demonstrated that sublancin destroyed and

343

perforated the cell surface of C. perfringens.14 In the present study, the electron

344

photomicrographs also revealed morphological damages caused by sublancin

345

treatment in MRSA cell structure. The presence of cell wall ruptures and cellular lysis,

346

in electron micrographs of MRSA ATCC43300 treated with 0.25 × MIC sublancin for

347

24 h, suggested that the antimicrobial action of sublancin involved the destruction of

348

the microbial cell wall leading to the escape of intracellular contents. In addition, the

349

susceptibility of S. aureus toward sublancin has been reported to rely on the presence 17



350

of large mechanosensitive channels, which might be prevented from closing, resulting

351

in leakage of essential metabolites from these cells.13 This natural antimicrobial

352

property imparts certain advantages to AMPs as compared to traditional antibiotics

353

that usually act by a defined high-affinity antibacterial target. It would be more

354

difficult for bacteria to develop resistance against a compound having a mode of

355

action that is physical in nature.40 Interestingly, an increased number of cells

356

containing whole or incomplete septa, after exposure to 0.25 × MIC sublancin for 1 h,

357

indicates that cell division of MRSA was almost completely inhibited. Similar effects

358

on cell division have been described for the lipopeptide daptomycin41 and the

359

lipoglycopeptide oritavancin.42 Cytotoxicity toward eukaryotic cells is a concern

360

during the development of AMPs. In this study, sublancin did not display cytotoxic

361

activity on RAW264.7 macrophage cells, mouse peritoneal macrophages, and human

362

Caco-2 epithelial cells even at a relatively high concentration.

363

Our previous study revealed that sublancin administered in the delayed-treatment

364

condition (6 hours after infection) has a good protective effect against sepsis induced

365

by methicillin sensitive S. aureus.15 The results obtained with in vitro assays prompted

366

us to exploit the in vivo potential of sublancin against MRSA challenge in mice. The

367

therapeutic effect of sublancin was initially investigated in a sub-lethal MRSA sepsis

368

model. We found that sublancin significantly reduced the bacterial burden, which was

369

similar to the efficacy of vancomycin. In an early infection phase (i.e., 24 hours

370

post-infection), sublancin tended to trigger the production of cytokines IL-6 and

371

MCP-1. MCP-1 is the primary chemokine that recruit monocytes. Therefore, 18


Page 18 of 41

Page 19 of 41


372

monocytes may be critical for the protective effect of sublancin. However, sublancin

373

substantially decreased TNF-α, IL-6, and MCP-1 levels 72 h after MRSA infection,

374

which indicated that sublancin could help to balance the immune response during

375

infection.

376

We then evaluated the protective capacity of sublancin in a lethal MRSA sepsis

377

model, and found that sublancin had an excellent protective activity for reducing

378

mortality and weight loss. Sepsis causes immunosuppression, increased gut and

379

immune apoptosis as well as organ disfunction.43,

380

regarded as the “motor” of the systemic inflammatory response with obvious

381

alterations in gut integrity observed in a variety of infections including MRSA.45, 46 In

382

the current study, MRSA challenge seriously damaged the jejunal villus structure,

383

which is consistent with our previous report.15 However, our results demonstrated that

384

sublancin ameliorated intestinal histopathological lesions and protected the intestinal

385

villi integrity of MRSA challenged mice. Higher sublancin treatment (4.0 mg/kg)

386

even helps villi to recover its original height. Similar phenomenon was reported by

387

Wang et al.14 who observed that sublancin significantly reduced the severity of

388

intestinal lesion of broilers challenged by C. perfringens. PCNA, a marker of cell

389

proliferation, was increased in the sublancin treatment (4.0 mg/kg). The proliferation

390

of intestinal cells facilitates nutrient absorption which is beneficial for the recovery of

391

MRSA challenged mice. NF-κB is a major regulator of inflammatory responses and

392

induced the expression of iNOS, an inflammatory marker.34 In the present study,

393

sublancin treated mice showed a decrease in NF-κB and iNOS production compared

44

The intestine has often been

19



394

with the MRSA group, which suggested that sublancin might attenuate the intestinal

395

inflammation by inhibition of the NF-κB activation.

396

Caspase-3, a potent inducer of apoptosis, was significantly increased in the

397

spleen due to the MRSA infection. In agreement with the current study, caspase-3

398

activation was also seen in S. aureus abscesses. We observed that sublancin decreased

399

the caspase-3 density in the spleen, which may be due to its ability to reduce the

400

bacterial burden. Additionally, MRSA challenge increased CD4+ T cell density in the

401

spleen but had no significant impact on CD8+ T cells, which was in agreement with

402

the findings of Chan et al.47 Sublancin prevented major activation of CD4+ cells

403

induced by MRSA, indicating that mice treated with sublancin did not develop the

404

same degree of activation of T helper cells. CD8+ T cells play a pivotal role in

405

elimination of infected cells by activating affected cells and cytotoxic effects.48 It is

406

reported that CD8+ T cells are key mediators of adaptive immunity against protozoan,

407

viral, and bacterial pathogens.49 The mechanism of sublancin in promoting CD8+ T

408

cells proliferation remains to be established.

409

In conclusion, our study indicates that the protection of sublancin in the mouse

410

intraperitoneal infection model is dependent on both its direct antibacterial activity

411

and its immunomodulatory properties. We posit that sublancin is a promising

412

therapeutic alternative for treating MRSA infections though further work is necessary

413

to successfully translate sublancin to the clinic. Moreover, this research provides

414

further vision into the development of naturally derived AMPs as immune therapy

415

regimens against drug-resistant pathogens. 20


Page 20 of 41

Page 21 of 41


416

AUTHOR INFORMATION

417

Corresponding Author

418

*Telephone:

419

[email protected].

420

Funding

421

This project was supported by the Special Fund for Agro-scientific Research in the

422

Public Interest (201403047) and National Key Research and Development Program of

423

China (2016YFD0501308).

424

Notes

425

The authors declare no competing financial interest.

+86-10-62731456.

Fax:

+86-10-62733688.

21


E-mail:


426

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574 575 576 577

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578

28


Page 28 of 41

Page 29 of 41

579


Table 1. Effects of sublancin on the body weight and cumulative mortality in MRSA ATCC43300 infected mice (n = 20 mice/group) Days after infection 0

1

2

3

Control

0

0

0

0

MRSA

0

10

35

65

MRSA + 0.5 mg/kg sublancin

0

0

20

35


0

0

10

25


0

0

5

15


0

0

0

10

Control

18.7 ± 0.44

20.1 ± 0.40

21.3 ± 0.47

22.9 ± 0.59

MRSA

18.7 ± 0.38

17.1 ± 0.57**

18.0 ± 0.53**

19.2 ± 0.62**


18.8 ± 0.31

18.4 ± 0.39**

19.1 ± 0.49**

20.2 ± 0.65**


18.6 ± 0.31

18.3 ± 0.38**

19.2 ± 0.43**

20.2 ± 0.51**


18.5 ± 0.37

18.6 ± 0.47**

19.7 ± 0.51**,#

20.6 ± 0.63**,#


18.7 ± 0.32

18.9 ± 0.50**

19.9 ± 0.52**,#

21.4 ± 0.38**,##

Cumulative mortality（%）

Body weight (g)

29



Page 30 of 41

580

Body weight data are presented as mean ± SD. Cumulative mortality is shown as the cumulative percentage of mice deaths relative to the total number of

581

mice treated at each time point. Compared with the control group, statistical significance is shown with *(p < 0.05), or **(p < 0.01). Compared with the

582

MRSA group, statistical significance is shown with #(p < 0.05), or ##(p < 0.01).

30


Page 31 of 41


583

Figure Legends

584

Figure 1. Effect of sublancin on the growth and cell morphology of MRSA. (A)

585

Photo of inhibition zone of MRSA ATCC43300 treated with and without sublancin. (1)

586

was control of MRSA. (2) and (3) were MRSA treated with 60 µM sublancin. (B)

587

Time-kill curves for MRSA ATCC43300 treated with sublancin (0.125-, 0.25-,

588

0.5-/1.0- fold MIC). (C) Scanning Electron Microscopy photomicrographs of MRSA

589

ATCC43300 without (1) and with (2) 0.25 × MIC sublancin at 24 h. (D) The effect of

590

sublancin on the structure of MRSA ATCC43300 cells as seen by Transmission

591

Electron Microscopy. (1) Untreated MRSA cells. (2–4) MRSA cells were incubated

592

with 0.25 × MIC sublancin for 0.5, 1 and 24 h respectively. (E) Effect of sublancin on

593

the percentage of MRSA cells containing septal components.

594

Figure 2. Lack of peptide toxicity. Assays evaluating the cytotoxic activity of

595

sublancin against RAW264.7 macrophages (A), mouse peritoneal macrophage (P-Mac)

596

(B) and Caco-2 cells. Cells were exposed to various concentrations of sublancin as

597

indicated for 24 h. Cell viability was evaluated using the cell counting kit-8 (CCK-8)

598

reagent, n = 6.

599

Figure 3. Efficacy of sublancin in MRSA-induced sub lethal infection model. (A)

600

Sublancin (2.0 mg/kg) was administered by intraperitoneal injection at -24, -6, 0, +3,

601

or +6 h after intraperitoneal inoculation of MRSA ATCC43300. The bacterial load

602

was measured by counting bacteria in the peritoneal lavage 24 hours after infection.

603

Values are expressed as the mean ± SEM, #p < 0.05,

##

p < 0.01 and

31


###

p < 0.001


Page 32 of 41

604

compared with that of vehicle group without sublancin treatment. (B) Mice were

605

treated intraperitoneally with a single dose of sublancin (2.0 mg/kg), vancomycin (2.0

606

mg/kg) and sterile saline 6 h after infection. Mice were analyzed for bacterial counts

607

in the peritoneal lavage at 24 and 72 h of infection. Values are expressed as the mean

608

± SEM, #p < 0.05 and

609

therapeutic treatment.

610

Figure 4. Effect of sublancin on the release of cytokines into the peritoneal cavity of

611

mice infected with MRSA. Mice were injected intraperitoneally with 1.0 × 108 CFU

612

of MRSA ATCC43300. Six hours after inoculation, sublancin (2.0 mg/kg),

613

vancomycin (2.0 mg/kg) or sterile saline was administered by intraperitoneal injection.

614

TNF-α, IL-6, and MCP-1 were analyzed in the peritoneal lavage at 24 and 72 h of

615

infection. Values are expressed as the mean ± SEM (n = 6), *p < 0.05, **p < 0.01, and

616

***p < 0.001 compared with that of control group. #p < 0.05,

617

0.001 compared with that of MRSA group.

618

Figure 5. The effect of sublancin on jejunal morphology of mice challenged with

619

MRSA. (A) Jejunal villi height in response to MRSA challenge and sublancin

620

treatment. (B) Immune staining of the jejunum for PCNA. (a) control, (b) MRSA

621

group, and (c) 4.0 mg/kg sublancin. The bar represents 50 µm. (C) Density of PCNA+

622

cells in the jejunal villi. Values are expressed as the mean ± SEM (n = 6), *p < 0.05

623

and **p < 0.01 compared with that of control group. ##p < 0.01 compared with that of

624

MRSA group.

##

p < 0.01 compared with that of MRSA challenge without

32


##

p < 0.01, and

###

p

Use of the Antimicrobial Peptide Sublancin with Combined

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